This project aims to develop, improve, and exploit new molecules, mostly genetically targetable, for measuring and/or manipulating neuronal messengers and signals such as calcium, cGMP, thiol-disulfide redox potential, glutamate, membrane potential, gene expression, and protein-protein interaction. Calcium indicators based on green fluorescent protein (GFP) mutants or small-molecule fluorophores will be genetically targeted to the sites such as the mouths of Ca2+ -conducting channels where privileged microdomains of Ca2+ have long been postulated but not directly measured. Transgenic animals expressing genetically targetable Ca24 indicators will facilitate analysis of mutants in Ca2+ -handling proteins and imaging of neuronal firing patterns. New indicators of cGMP based on GFP mutants fused to cGMP dependent protein kinase will be improved and used to visualize spatiotemporal dynamics of cGMP, especially during synaptic plasticity. Kinetics and specificity of new GFP-based indicators of thiol-disulfide redox potential will be characterized so that redox changes during channel modulation, growth stimulation, and cell death can be imaged. Attempts will be made to develop GFP-based indicators of glutamate to enable imaging of the location and amplitude of extracellular glutamate transients during synaptic plasticity and excitotoxicity. New small molecules will be synthesized to overcome the most important existing limitations in measurement of membrane potential and gene expression by fluorescence resonance energy transfer. Proteins that could mediate light-activated depolarization or hyperpolarization will be transfected into mammalian cells, eventually neurons, to permit combined genetic and optical control of excitability. Improvements are sought in four generic technologies that could greatly facilitate construction of genetically encoded indicators or in vivo analysis of protein function: red fluorescent proteins from coral, targeting of biarsenical dyes to small protein motifs containing four cysteines, targeting of small zinc ligands to motifs containing multiple histidines, and assays to measure distances of 2 to 50 nm between pairs of endogenous unlabeled proteins within fixed tissues, using fluorescence detection but not energy transfer. Such new techniques should help explore the biochemical mechanisms of long-term depression and a newly discovered form of long-term potentiation of synapses between parallel fibers and Purkinje neurons in young adult cerebellar slices.